Journal: PLoS ONE

Article Title: On the influence of cannabinoids on cell morphology and motility of glioblastoma cells

doi: 10.1371/journal.pone.0212037

Figure Lengend Snippet: A) shows the mean speed and standard error of the mean (sem) of single LN229, U87 and U138 cells, when treated with cannabinoid receptor inverse agonists. B) and C) depict the directionality and contact area of the same cell lines and treatments together with the sem. For all parameters cell line specific changes that have no apparent receptor specificity can be observed. Statistics was performed using a Kruskal-Wallis test and significance was chosen for p<0.05. The asterisk denotes significant results regarding the respective measurement indicated with the bar.

Article Snippet: U87 and LN229 cells were purchased from the American Type Culture Collection (Manassas, VA, USA; U87: ATCC HTB-14; LN229: ATCC CRL-2611) and U138 cells were obtained from Cell Lines Service (Cell Lines Service, 300363).

Techniques:

Journal: PLoS ONE

Article Title: On the influence of cannabinoids on cell morphology and motility of glioblastoma cells

doi: 10.1371/journal.pone.0212037

Figure Lengend Snippet: A) shows the mean circularity and sem of single LN229, U87 and U138 cells, when treated with cannabinoid receptor inverse agonists. B) and C) depict the apparent brightness and homogeneity of the same cell lines and treatments together with the sem. For all parameters cell line specific changes that have no apparent receptor specificity can be observed. Statistics was performed using a Kruskal-Wallis test and significance was chosen for p<0.05. The asterisk denotes significant results regarding the respective measurement indicated with the bar.

Article Snippet: U87 and LN229 cells were purchased from the American Type Culture Collection (Manassas, VA, USA; U87: ATCC HTB-14; LN229: ATCC CRL-2611) and U138 cells were obtained from Cell Lines Service (Cell Lines Service, 300363).

Techniques:

Journal: PLoS ONE

Article Title: On the influence of cannabinoids on cell morphology and motility of glioblastoma cells

doi: 10.1371/journal.pone.0212037

Figure Lengend Snippet: A) shows the correlation between apparent brightness and homogeneity of the three cell lines, with an correlation coefficient of r = 0.848 [0.831;0.863]. B), C), D), E) and F) illustrate the correlation between the single cell parameters brightness and contact area, homogeneity and contact area, circularity and contact area, brightness and circularity as well as cell speed and circularity, with correlations coefficients of r = -0.576 [-0.612;-0.536], r = -0.456 [-0.500;-0.410], r = -0.480 [-0.522;-0.435], r = 0.516 [0.473;0.556] and with r = -0.122 [-0.177; 0.066], respectively. Red dots correspond to LN229 cells, blue ones to U138 cells and green dots to U87 cells. The black line corresponds to the respective linear fit.

Article Snippet: U87 and LN229 cells were purchased from the American Type Culture Collection (Manassas, VA, USA; U87: ATCC HTB-14; LN229: ATCC CRL-2611) and U138 cells were obtained from Cell Lines Service (Cell Lines Service, 300363).

Techniques: Single Cell

Journal: PLoS ONE

Article Title: On the influence of cannabinoids on cell morphology and motility of glioblastoma cells

doi: 10.1371/journal.pone.0212037

Figure Lengend Snippet: A) depicts the phalloidin (green) and DAPI (blue) staining of LN229 control cells on the left and the respective structure image of the actin cytoskeleton as a heat map on the right. A correspondence of highly structured regions in the actin staining with the respective structure image is visible. Furthermore, unstructured, homogeneous regions do not contribute to the structure image, as for example in the center of the image. The scaling corresponds to 20 μm. B) shows the quantification of the density of actin structures for U87 cells treated with cannabinoid receptor agonists and inverse agonists as mean value with the sem. C) and D) illustrates the density of actin structures in LN229 and U138 cells after cannabinoid treatment. Various effects that show no apparent receptor specificity were observed. Statistics was performed using a Kruskal-Wallis test and significance was chosen for p<0.05. The asterisk denotes significant results regarding the respective measurement indicated with the bar.

Article Snippet: U87 and LN229 cells were purchased from the American Type Culture Collection (Manassas, VA, USA; U87: ATCC HTB-14; LN229: ATCC CRL-2611) and U138 cells were obtained from Cell Lines Service (Cell Lines Service, 300363).

Techniques: Staining, Control

Journal: Translational Cancer Research

Article Title: The LINC00957/miR-17-5p axis regulates the cell cycle and migration in glioblastoma via the cuproptosis-related gene nephronectin

doi: 10.21037/tcr-24-450

Figure Lengend Snippet: Knocking down NPNT enhances the sensitivity of glioma cells to cuproptosis inducers. (A) The CCK8 assay of U87 and LN229 cells treating with elesclomol. (B) The ROS levels of shNC or shNPNT U87 and LN229 cells treating with elesclomol and CuCl2. (C) The Copper concentration of shNC or shNPNT U87 and LN229 cells treating with different concentration of elesclomol (10 and 20 nM). Data were expressed as the mean ± standard deviation of three independent experiments (n=3). ns, not significant; ****, P<0.0001. IC 50 , half-maximal inhibitory concentration; ROS, reactive oxygen species; NPNT, nephronectin; CCK8, Cell Counting Kit-8; shNC, shRNA negative control; shNPNT, shRNA NPNT; shRNA, short hairpin RNA.

Article Snippet: U87 and LN229 cells were cultured in 6 cm plates for 24 h and treated with a cuproptosis inducers (elesclomol) for 24 h. Following this, the cells were collected and disrupted ultrasonically to detect intracellular copper according to the manufacturer’s instructions (E-BC-K775-M, Elabscience, Houston, TX, USA).

Techniques: CCK-8 Assay, Concentration Assay, Standard Deviation, Cell Counting, shRNA, Negative Control

Journal: Translational Cancer Research

Article Title: The LINC00957/miR-17-5p axis regulates the cell cycle and migration in glioblastoma via the cuproptosis-related gene nephronectin

doi: 10.21037/tcr-24-450

Figure Lengend Snippet: LncRNA LINC00957 and miR-17-5p can regulate NPNT protein in GBM tumors. (A) ROC curve of the AL365361.1, AL591848.3, LINC00957, and AF111167.2. (B) NPNT mRNA levels and AL365361.1, AL591848.3, LINC00957, and AF111167.2 in LN229 cells with ASO knockdown four potential lncRNAs was detected. Data were expressed as the mean ± standard deviation of three independent experiments (n=3). ns, not significant; ***, P<0.001; ****, P<0.0001. AUC, area under the curve; ASO, antisense oligonucleotide; NC, negative control; lncRNA, long non-coding RNA; NPNT, nephronectin; GBM, glioblastoma; ROC, receiver operating characteristic; mRNA, messenger RNA.

Article Snippet: U87 and LN229 cells were cultured in 6 cm plates for 24 h and treated with a cuproptosis inducers (elesclomol) for 24 h. Following this, the cells were collected and disrupted ultrasonically to detect intracellular copper according to the manufacturer’s instructions (E-BC-K775-M, Elabscience, Houston, TX, USA).

Techniques: Knockdown, Standard Deviation, Negative Control

Journal: Translational Cancer Research

Article Title: The LINC00957/miR-17-5p axis regulates the cell cycle and migration in glioblastoma via the cuproptosis-related gene nephronectin

doi: 10.21037/tcr-24-450

Figure Lengend Snippet: The LINC00957, miR-17-5p, and NPNT regulate the malignant progression of GBM. (A) The GO functional analysis of the related biological processes of DEGs in low expression group and high expression group among LINC00957 and NPNT. (B) The CCK8 results in U87 cells with knockdown of LINC00957 and NPNT. (C) The transwell results of crystal violet staining at 40× magnification in LN229 cells with knockdown of LINC00957. (D) The cell cycle results in LN229 cells with knockdown of NPNT. Data were expressed as the mean ± standard deviation of three independent experiments (n=3). ns, not significant; ****, P<0.0001. ASO, antisense oligonucleotide; NC, negative control; CCK8, Cell Counting Kit-8; OD450, optical density at 450 nm; shNC, shRNA negative control; shNPNT, shRNA NPNT; shRNA, short hairpin RNA; NPNT, nephronectin; GBM, glioblastoma; GO, gene ontology; DEG, differentially expressed gene.

Article Snippet: U87 and LN229 cells were cultured in 6 cm plates for 24 h and treated with a cuproptosis inducers (elesclomol) for 24 h. Following this, the cells were collected and disrupted ultrasonically to detect intracellular copper according to the manufacturer’s instructions (E-BC-K775-M, Elabscience, Houston, TX, USA).

Techniques: Functional Assay, Expressing, Knockdown, Staining, Standard Deviation, Negative Control, Cell Counting, shRNA

Journal: Translational Cancer Research

Article Title: The LINC00957/miR-17-5p axis regulates the cell cycle and migration in glioblastoma via the cuproptosis-related gene nephronectin

doi: 10.21037/tcr-24-450

Figure Lengend Snippet: The lncRNA LINC00957 and miR-17-5p can regulate NPNT protein in GBM tumors. (A) Correlation analysis between NPNT, LINC00957, and miR-17-5p. (B) The RNA levels of NPNT, LINC00957, and miR-17-5p in U87 and LN229 with ASO knockdown LINC00957 was detected; the RNA levels of NPNT, LINC00957 and miR-17-5p in U87 and LN229 treated with miR-17-5p mimic was detected; the RNA levels of NPNT, LINC00957, and miR-17-5p in U87 and LN229 treated with miR-17-5p inhibitor was detected. (C) Predicted binding site between LINC00957 and miR-17-5p. (D) Luciferase reporter gene test was used to test the relationship between LINC00957 and miR-17-5p treated with miR-17-5p mimic in U87 cell. (E) The CCK8 results in ASO NC or ASO LINC00957 U87 cells treated with shNC or shNPNT. Data were expressed as the mean ± standard deviation of three independent experiments (n=3). ns, not significant; ****, P<0.0001. NPNT, nephronectin; ASO, antisense oligonucleotide; NC, negative control; WT, wild-type; MUT, mutated; shNC, shRNA negative control; shNPNT, shRNA NPNT; shRNA, short hairpin RNA; lncRNA, long non-coding RNA; GBM, glioblastoma; CCK8, Cell Counting Kit-8.

Article Snippet: U87 and LN229 cells were cultured in 6 cm plates for 24 h and treated with a cuproptosis inducers (elesclomol) for 24 h. Following this, the cells were collected and disrupted ultrasonically to detect intracellular copper according to the manufacturer’s instructions (E-BC-K775-M, Elabscience, Houston, TX, USA).

Techniques: Knockdown, Binding Assay, Luciferase, Standard Deviation, Negative Control, shRNA, Cell Counting

Journal: American Journal of Cancer Research

Article Title: Claudin 4 enhances the malignancy of glioma cells via NNAT/Wnt signaling

doi:

Figure Lengend Snippet: CLND4 promotes malignancy in glioma cells. A. Expression levels (mRNA) of CLDN4 in normal tissues (n=5) and glioma tissues (n=156); data derived from TCGA database. B. Representative images of immunohistochemical staining for CLDN4 in para-carcinomatous tissues (n=15), early-stage glioma tissues (stage I-II, n=15), and late-stage glioma tissues (stage III-IV, n=15); scale bar =50 μm. C. Kaplan-Meier survival curve for high and low expression of CLDN4 in glioma patients (n=513); data from TCGA database. D. Western blotting for CLDN4 in LN229/T98G cells transfected with empty vector or a CLDN4-overexpression vector. E. Cell proliferation assay (CCK-8 assay) for LN229/T98G cells transfected with empty vector or CLDN4-overexpression vector. T98G, P<0.05, 72 hours; LN229, P<0.01, 72 hours. F. Cell migration assay (Transwell assay) for LN229/T98G cells transfected with empty vector or CLDN4-overexpression vector; scale bar =100 μm. G. Colony formation assay for LN229/T98G cells transfected with empty vector or CLDN4-overexpression vector.

Article Snippet: Transfected LN229 and T98G cells overexpressing CLDN4 were obtained from GenScript ProBio (China); the same cells transfected with the empty vector (without the CLDN4 gene) were also obtained from the same source.

Techniques: Expressing, Derivative Assay, Immunohistochemical staining, Staining, Western Blot, Transfection, Plasmid Preparation, Over Expression, Proliferation Assay, CCK-8 Assay, Cell Migration Assay, Transwell Assay, Colony Assay

Journal: American Journal of Cancer Research

Article Title: Claudin 4 enhances the malignancy of glioma cells via NNAT/Wnt signaling

doi:

Figure Lengend Snippet: CLDN4-stimulated NNAT upregulation. A. The top 30 upregulated genes in patients with gliomas that had high CLDN4 expression levels as compared to those with gliomas having low CLDN4 expression levels; data derived from TCGA database (n=513). B. Western blotting for CLDN4 and NNAT in LN229/T98G cells transfected with empty vector or CLDN4-overexpression vector. C. Cell proliferation assays for LN229/T98G cells transfected with empty vector or CLDN4-overexpression vector treated with scramble or NNAT siRNAs. LN229, vector vs vector + siRNA, P>0.05, OE vs OE + siRNA, P<0.05, 72 hours; T98G, vector vs vector + siRNA, P>0.05, OE vs OE + siRNA, P<0.01, 72 hours. D. Cell migration assay for LN229/T98G cells transfected with empty vector or CLDN4-overexpression vector treated with scramble or NNAT siRNAs. E. Colony formation assays for LN229/T98G cells transfected with empty vector or CLDN4-overexpression vector treated with scramble or NNAT siRNAs. F. Representative images of immunohistochemical staining for CLND4 in early-stage glioma tissues (stage I-II, n=15) and late-stage glioma tissues (stage III-IV, n=15); scale bar =100 μm.

Article Snippet: Transfected LN229 and T98G cells overexpressing CLDN4 were obtained from GenScript ProBio (China); the same cells transfected with the empty vector (without the CLDN4 gene) were also obtained from the same source.

Techniques: Expressing, Derivative Assay, Western Blot, Transfection, Plasmid Preparation, Over Expression, Cell Migration Assay, Immunohistochemical staining, Staining

Journal: American Journal of Cancer Research

Article Title: Claudin 4 enhances the malignancy of glioma cells via NNAT/Wnt signaling

doi:

Figure Lengend Snippet: The CLDN4/NNAT axis modulates glioma progression through Wnt signaling. (A and B) The results of the KEGG enrichment analysis for the major signaling pathways involved in glioma progression in 513 glioma patients divided into CLDN4-high/low (A) or NNAT-high/low (B) groups. (C) Western blotting for Wnt1, Wnt2, and Wnt3A in LN229 cells transfected with empty vector or CLDN4-overexpression vector. (D) Western blotting for Wnt3A in LN229/T98G cells transfected with empty vector or CLDN4-overexpression vector treated with scramble or NNAT siRNAs. (E) Cell proliferation assays for LN229/T98G cells transfected with empty vector or CLDN4-overexpression vector treated with PBS or pyrvinium pamoate (10 nM). LN229, vector vs vector + PP, P>0.05, OE vs OE + PP, P<0.05, 72 hours; T98G, vector vs vector + PP, P>0.05, OE vs OE + PP, P<0.05, 72 hours. (F) Cell migration assays for LN229/T98G cells transfected with empty vector or CLDN4-overexpression vector treated with PBS or pyrvinium pamoate (10 nM). (G) Colony formation assays for LN229/T98G cells transfected with empty vector or CLDN4-overexpression vector treated with PBS or pyrvinium pamoate (10 nM).

Article Snippet: Transfected LN229 and T98G cells overexpressing CLDN4 were obtained from GenScript ProBio (China); the same cells transfected with the empty vector (without the CLDN4 gene) were also obtained from the same source.

Techniques: Protein-Protein interactions, Western Blot, Transfection, Plasmid Preparation, Over Expression, Migration

Journal: American Journal of Cancer Research

Article Title: Claudin 4 enhances the malignancy of glioma cells via NNAT/Wnt signaling

doi:

Figure Lengend Snippet: CLDN4 facilitates glioma growth in vivo. A. Western blotting for CLDN4 in primary glioma cells derived from 6 patients. B. Representative images of PDOs derived from CLDN4-high and -low glioma tissues; scale bar =50 μm. C. Western blotting for Wnt3A and NNAT in PDOs derived from CLDN4-high and -low glioma tissues. D. Representative images for immunofluorescence staining for Ki67 in PDOs derived from CLDN4-high and -low glioma tissues; scale bar =50 μm. E. Tumor volumes of subcutaneous tumors formed by LN229 cells transfected with empty vector or CLDN4-overexpression vector (n=5 in each group). F. Kaplan-Meier survival curve for mice with tumors formed by LN229 cells transfected with empty vector or CLDN4-overexpression vector (n=5 in each group).

Article Snippet: Transfected LN229 and T98G cells overexpressing CLDN4 were obtained from GenScript ProBio (China); the same cells transfected with the empty vector (without the CLDN4 gene) were also obtained from the same source.

Techniques: In Vivo, Western Blot, Derivative Assay, Immunofluorescence, Staining, Transfection, Plasmid Preparation, Over Expression

Journal: Frontiers in Oncology

Article Title: System analysis based on the migration- and invasion-related gene sets identifies the infiltration-related genes of glioma

doi: 10.3389/fonc.2023.1075716

Figure Lengend Snippet: EMP3 silencing attenuates the migration and invasion of glioma cells. (A) The expression of EMP3 in different cell lines in the CCLE datasets. (B–D) Western blot and qPCR display EMP3 express highly in U118 and A172. (E–G) The growth curve and plate cloning experiment display that EMP3 silencing has little effect on the cell proliferation of U118 and A172. (H, I) EMP3 knockdown inhibits the migration and invasion of glioma cells. ( J–O , 20×) qPCR displays EMP3 silencing attenuates the expression of MMP2 and MMP9 expression (ns, p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).

Article Snippet: The normal human astroglia (NHA), which were purchased from the Chongqing Golden Magpie Technology Development Co., and U251, LN229, A172, U118, and U87 cells, which were purchased from the China Center for Type Culture Collection (Shanghai, China), were cultured in the DMEM medium supplemented with 10% FBS (HyClone, UT, USA) and 1% penicillin–streptomycin (Beyotime Biotechnology, Jiangsu, China).

Techniques: Migration, Expressing, Western Blot, Cloning, Knockdown

Journal: Frontiers in Oncology

Article Title: System analysis based on the migration- and invasion-related gene sets identifies the infiltration-related genes of glioma

doi: 10.3389/fonc.2023.1075716

Figure Lengend Snippet: EMP3 silencing suppressed EMT marker expression. (A) Western blot analysis of U118 transfected with indicated siRNAs targeting EMP3 (siEMP3#1, siEMP3#2, and siEMP3#3) or siNC. (B) Western blot analysis of A172 transfected with indicated siRNAs targeting EMP3 (siEMP3#1, siEMP3#2, and siEMP3#3) or siNC. (p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Article Snippet: The normal human astroglia (NHA), which were purchased from the Chongqing Golden Magpie Technology Development Co., and U251, LN229, A172, U118, and U87 cells, which were purchased from the China Center for Type Culture Collection (Shanghai, China), were cultured in the DMEM medium supplemented with 10% FBS (HyClone, UT, USA) and 1% penicillin–streptomycin (Beyotime Biotechnology, Jiangsu, China).

Techniques: Marker, Expressing, Western Blot, Transfection